Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments' images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for 3D microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting Essential Metadata for 3D BRAIN Microscopy includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration.

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